OPEN 3 ACCESS Freely available online
Dynamically-Driven Enhancement of the Catalytic A
Machinery of the SARS 3C-Like Protease by the S284-
T285-I286/A Mutations on the Extra Domain
Liangzhong Lim 1 ®, Jiahai Shi 19 ", Yuguang Mu 2 , Jianxing Song 1 *
1 Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore, 2 School of Biological Sciences, Nanyang
Technological University, Singapore, Republic of Singapore
Abstract
Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the
R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated
the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with
S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined
its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly
tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/
A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly
packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra
domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues,
which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances
the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation
analysis reveals that a global network of the correlated motions exists in the protease, whose components include all
residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally
decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present
study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which
is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite
catalytic effects. Our results thus imply a promising avenue to design specific inhibitors for 3CL proteases by disrupting their
dynamic correlation network.
@ PLOS | one
Citation: Lim L, Shi J, Mu Y, Song J (2014) Dynamically-Driven Enhancement of the Catalytic Machinery of the SARS 3C-Like Protease by the S284-T285-I286/A
Mutations on the Extra Domain. PLoS ONE 9(7): el 01 941. doi:10.1371/journal.pone.0101941
Editor: Franca Fraternali, King's College, London, United Kingdom
Received January 13, 2014; Accepted June 13, 2014; Published July 18, 2014
Copyright: © 2014 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by Ministry of Education of Singapore (MOE) Tier 3 Grant R-1 54-002-580-1 1 2, and also partly by Tier 2 Grant MOE 201 1 -T2-1 -
096 (R1 54-000-525-1 12) to Jianxing Song. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: dbssjx@nus.edu.sg
9 These authors contributed equally to this work.
n Current address: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America
Introduction
In 2002, severe acute respiratory syndrome (SARS) suddenly
broke out in China and then rapidly spread to 32 countries,
resulting in ~8500 infections and over 900 deaths (http:/ /www.
who.int/csr/ sars/ en/). It is the first emerging infectious disease of
the 21st century and was caused by a coronavirus termed SARS-
CoV. Although now SARS appears to be contained, new
coronaviruses have been detected, which may cause great threats
to the human health. For example, since the appearance of a new
coronavirus termed Middle East respiratory syndrome coronavirus
(MERS-CoV) in April 2012, it has caused 207 confirmed cases,
out of which 84 died (http://www.who.int/csr/disease/
coronavirus_infections/en/index.html). More importandy, so far
neither a vaccine nor an efficacious therapy has been available for
them. Therefore, it remains highly demanded to develop strategies
to design potential therapeutic agents against SARS- and other
CoVs.
Among the known RNA viruses, coronaviruses are enveloped,
positive-stranded ones with the largest single-stranded RNA
genome (27-31 kilobases). The large replicase gene encodes two
viral polyproteins, namely ppla (486 kDa) and pplab (790 kDa),
which have to be processed into active subunits for genome
replication and transcription by two viral proteases [1,2], namely
the papain-like cysteine protease (PL2pro) and 3C-Like protease
(3CLpro), also known as main protease (Mpro). Previously, SARS
3CLpro has been extensively characterized to be a key target for
development of antiviral therapies. The coronavirus 3CLpro is so
named to reflect the similarity of its catalytic machinery to that of
the picornavirus 3C proteases [1-3]. Noticeably, both 3C and
3CL-Like proteases utilize the two-domain chymotrypsin fold to
host the complete catalytic machinery, which is located in the cleft
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Dynamical Enhancement of SARS-CoV 3CLpro
between domains I and II. Intriguingly, however, in the
coronavirus 3CLpro, a ~100-residue helical domain was evolu-
tionarily acquired at its C-terminus [1-4]. Moreover, unlike 3C
protease, only the homodimeric form is catalytically competent for
the CoV 3CLpro [1,4,5-24]. After intense studies, now it has been
clear that both chymotrypsin fold and extra domain are critical for
dimerization.
We were particularly interested in understanding the role of the
extra domain and thus initiated a domain dissection study on
SARS 3CLpro immediately after the SARS outbreak in Singapore
[6] . The results revealed that although the catalytic fold and extra
domain could fold independendy, the catalytic fold alone was
monomeric and almost inactive. This indicates that the extra
domain plays a key role in maintaining the dimerization, thus
mediating the catalysis [6]. Therefore, we further conducted a
systematic mutagenesis study which led to identification of the
extra-domain residues critical for both dimerization and catalysis
[16]. Interestingly, we found that the residues important for
catalysis and dimerization constitute a nano-channel, which are
composed of residues from both catalytic and extra domains [16].
Moreover, we determined the high-resolution structure of R298A,
a monomeric mutant triggered by a point mutation on the extra
domain, in which the most radical changes have been found
within the catalytic machinery [22]. R298A adopts a completely
collapsed and inactivated catalytic machinery which is structurally
distinguishable from that in wild-type (WT) enzyme; with a short
3 10 -helix formed by residues Serl39-Phel40-Leul41 within the
oxyanion-binding loop [22]. Remarkably, the collapsed catalytic
machinery observed in R298A appears to represent a universal
inactivated state intrinsic to all inactive enzymes as the same
collapsed machinery was found in other monomers triggered by
the mutations G11A, N28A and S139A which are all located on
the chymotrypsin fold [21,23,24]. On the other hand, previously
we also identified a mutant N214A, which owns a dramatically
abolished activity but only slightly weakened dimerization [16].
Very unexpectedly, our determination of its crystal structure
revealed that it still adopts a dimeric structure almost identical to
that of the WT protease [25]. Nevertheless, the results with
molecular dynamics (MD) simulations up to 30 ns unveiled that
the N214A mutation triggers the dynamical instability of the
catalytic machinery, with many key residues jumped to sample the
conformations characteristic of the collapsed and inactivated state
in R298A, thus establishing a dynamically-driven inactivation
mechanism for the catalytic machinery of the SARS 3CLpro [25].
Here we studied another mutant we previously identified with
three residues S284-T285-I286 mutated to Ala, designated as
STI/A [16]. Interestingly, despite being far away from the active
pocket, the mutations led to a 3.6-fold enhancement of the
catalytic activity but only slighdy enhanced dimerization. Here,
our determination of its crystal structure reveals that STI/ A still
adopts the dimeric structure almost identical to that of the wild-
type (WT), only with a slighdy change of the extra-domain
packing, which is similar to that observed in the more active form
of the WT at high pH values (pH 7.6 and 8) [44]. As a
consequence, to understand its underlying dynamical mechanism,
we conducted 100-ns MD simulations for both WT and STI/A.
Remarkably, the most dramatic changes in STI/A simulations are
associated with the nano-channel. This change appears to slighdy
enhance the dynamic stability of the N-finger and helix A, which
subsequently relay the dynamic effects to the contacted residues
and finally to catalytic machinery. Ultimately, the key components
composed of the catalytic machinery become more dynamically
stable, thus rationalizing the enhancement of its catalytic activity.
As coronavirus 3C-Like proteases share a similar enzymatic
mechanism [1,2,26], our results would facilitate the development
of strategies and agents by modulating protein dynamics to fight
new coronaviruses whose outbreaks may occur in the future.
Materials and Methods
Accession Numbers
The structure coordinate of the STI/ A mutant was deposited in
Protein Data Bank, with PDB ID code of 3EA8.
Generation of the recombinant STI/A mutant without
any extra residue
Recently the extra N-terminal residues leftover from the
cleavage of fusion proteins were demonstrated to significandy
reduce the enzymatic activity [10,18]. On the other hand, the
SARS 3CLpro we previously studied had two extra residues Gly-
Ser after the thrombin cleavage of the GST-3CLp fusion proteins
[6,16]. In order to remove the two extra residues, in the present
study we transferred the gene encoding SARS 3CLp from the
pGEX-4T- 1 GST-fusion expression vector (Amersham Bioscienc-
es, GE Healthcare, Little Chalfont, UK) to the His-tagged pET28a
vector. Subsequendy, site-directed mutagenesis was utilized to
shorten the thrombin cleavage sequence LVPR | GS (CTG GTT
CCG CGT GGA TCC) engineered by the company to LVPR|
(CTG GTT CCG CGT), which only constituted the thrombin
cleavage site in conjunction with the first two N-terminal residues
Ser-Gly of SARS 3CLp. Interestingly, thrombin cleaved this new
site (LVPR | SG) very efficiently to release the authentic wild-type
3CLpro as well as N214A [25]. To produce STI/A mutant, site-
directed mutagenesis was further used to mutate S284-T285-I286
to Ala as previously described [16,25]. Benefited from this re-
engineered cleavage site, we were able to produce both authentic
WT 3CLpro [25] and its STI/A mutant without any extra
residues from the fusion tag.
Experimentally, the His-tagged protease or mutants was
expressed in E. coli strain BL21 (DE3) with induction by
0.4 mM isopropyl-l-thio-d-galactopyranoside (IPTG) under 20
degree overnight. The protease was obtained by in-gel cleavage
with thrombin while the His-tag protein attached to the Nickel-
NTA beads (QIAGEN), foUowed by a FPLC purification on a gel
filtration column (HiLoad 16/60 Superdex 200). The pure
protease was concentrated to 10 mg/ml and stored in the buffer
(10 mM Tris-HCl, 10 mM DTT, pH 7.5). The molecular weight
of the WT and N2 1 4A proteases were determined by a Voyager
STR MALDI-TOF mass spectrometer (Applied Biosystems).
Enzymatic activity assay and ITC characterization of
dimerization
The enzymatic activities of the STI/A proteases were
measured by a fluorescence resonance energy transfer (FRET)-
based assay using a fluorogenic substrate peptide (Dabcyl-
KTSAVLQSGFRKME-Edans) as previously described
[15,16,25,27]. Briefly, 1 ml reaction mixture contained 50 nM
protease and fluorogenic substrate with concentrations ranging
from 1 uM to 40 uM in a 5 mM Tris-HCl buffer with 5 mM
DTT at pH 6.0, which is identical to the pH for crystallization.
The enzyme activity was measured by monitoring the increase of
the emission fluorescence at a wavelength of 538 nm with
excitation at 355 nm using a Perkin-Elmer LS-50B luminescence
spectrometer. The Km and kcat values were deduced from data
analysis using Graphpad prism.
ITC (isothermal titration calorimetry) experiments were carried
out to determine the monomer-dimer dissociation constants of the
STI/A proteases as previously described [10,25] using a Microcal
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Dynamical Enhancement of SARS-CoV 3CLpro
VP ITC machine. Briefly, the protease samples and buffers were
span at 13.3k rpm for one hour to remove the tiny particles and
degas thoroughly. In titrations, the STI/A sample in 5 mM Tris-
HC1 buffer at pH 6.0 containing 5 mM DTT were loaded in the
syringe, which was subsequently titrated into the same buffer in
the cell. The obtained titration data with endothermic peaks were
analyzed by the built-in Microcal ORIGIN software using a
dimer-monomer dissociation model to generate the dissociation
constants and the enthalpy changes.
Crystallization, structure determination of the STI/A
protease
The STI/A protease with a concentration of 10 mg/ml were
crystallized in a 2 (0.1 hanging drop using a condition identical to
that previously reported except for a minor variation of the
PEG6000 concentration [4,18,25]. After a three-day growth, a
single crystal was picked up from the crystal clusters for diffraction
with the cryoprotective buffer (20% glycerol with the mother
liquid). The X-ray diffraction data were collected at Bruker X8
PROTEUM in-house X-ray machine.
The collected data sets were processed using the program
HKL2000 in a resolution of 2.25 A in the space group C2. Briefly,
the phase determination for the mutant structure was done by the
molecular replacement method using the WT SARS-CoV 3CLpro
structure (PDB code: 2H2Z) [18] as the searching model by the
program Phaser [28] in the program suite Phenfx [29]. The Ala
mutating residues (Ser284, Thr285, Ile286) were corrected in the
program COOT [30] . The refinements and the addition of the
solvent molecules of the models for the mutant were done in the
program suite Phenix. The final model was analyzed by
PROCHECK [31] and all figures were prepared using Pymol
[32].
Molecular dynamics (MD) simulations
The crystal structures of the WT (PDB code: 2H2Z) [18] and
STI/A determined in the present study were selected as the initial
models for molecular dynamics simulations as previously described
[25]. The crystal structures PDB files were post-processed as
previously described [33,34].
The simulation cell is a periodic cubic box with a minimum
distance of 10 A between the protein and the box walls to ensure
the protein would not directiy interact with its own periodic
images given the cutoff. The water molecules, described using the
TIP3P model, were filled in the periodic cubic box for the all atom
simulation, 6 Na + ions were randomly placed to neutralize the
charge in MD system for STI/A and WT dimers, Each system
contained approximately 75,000 atoms.
Three independent 100-ns MD simulations for each protease
were performed with the program GROMAGS [35] with the
AMBER-03 [36] all-atom force field. The long-range electrostatic
interactions were treated using the fast particle-mesh Ewald
summation method [35], with the real space cutoff of 9 A and a
cutoff of 14 A was used for the calculation of van der Waals
interactions. The temperature during the simulations was kept
constant at 300 K by Berendsen's coupling. The pressure was held
at 1 bar. The isothermal compressibility was 4.5*10~ 5 bar" 1 . The
time step was set as 2 fs. All bond lengths including hydrogen
atoms were constrained by the LINCS algorithm [37]. Prior to
MD simulations, all the initial structures were relaxed by 500 steps
of energy minimization using the steepest descent algorithm,
followed by 100 ps equilibration with a harmonic restraint
potential applied to all the heavy atoms of the protease.
Calculation of enclosed volume
POVME program was used to calculate the enclosed volumes of
the nano-channel and Thr25-Cys44 Leu-P2 substrate pocket [38].
Snapshots from each MD trajectory were taken at every 100-ps
interval. A 3D-grid spacing was constructed around the C p of
residues: Ser284, Thr285, Ile286 in the nano-channel, and Thr25
and Cys44 in the Leu-P2 substrate pocket. Grid points were
included when they do not overlap with any atom of the studied
residues or neighboring residues. The grid points were summed up
to give the enclosed volume.
Calculation of center of mass (COM), angle ©
Heavy atoms of all atoms are used in the computation of center
of mass: C, N, O and S. Hydrogen atoms are ignored. The COM
is calculated at lps interval. For the angle 0 calculation, COM of
the two chymotrypsin fold (residues 7-180) of both protomers and
the extra-domain/Domainlll (residue 200-302) of each protomer.
A vector is computed for each COM of Domain III to the COM
of the 2 chymotrypsin-folds. The angle © is the angle between
these 2 vectors.
Correlation analysis
STI/A as well as N214A modulates the catalysis of the SASR
3CL protease without substantial conformational change, suggest-
ing that the mutation effects are relayed to the catalytic machinery
through dynamic allostery. So here we attempted to analyze the
allosteric networks of WT, N214A and STI/A by a recendy
established approach called Mutlnf [39]. Mutlnf represents an
entropy-based approach to analyze ensembles of protein con-
formers, such as those from molecular dynamics simulations by
using internal coordinates and focusing on dihedral angles. In
particular, this approach is even applicable in cases for which
conformational changes are subtie, for example, because the
coupling is mostiy entropic in nature [39]. Briefly, this approach
utilizes second-order terms from the configurational entropy
expansion, called the mutual information, to identify pairs of
residues with correlated conformations, or correlated motions, in
an equilibrium ensemble [39]. In the present study, the
normalized matrix values were used, and 0.3 was set up to be
the threshold value to determine the pairs of highly correlated
residues. The residue pairs with correlated values &0.3 make up
the top 1% of the entire matrix.
Results
Activity and dimerization of the STI/A enzyme
By reengineering the thrombin cleavage site, we succeeded in
obtaining the STI/A proteases without any extra residues leftover
from the fusion tag. The enzyme was characterized by far-UV CD
spectroscopy and its spectrum (spectra not shown) had no
detectable difference from that with two extra residues Gly-Ser
that we previously studied [16], thus indicating that the two extra
residues have no detectable effect on the secondary structures.
By a fluorescence resonance energy transfer (FRET)-based
assay, we have measured the enzymatic activities of the authentic
STI/A proteases. As shown in Figure la, the STI/A protease has
a Km value (23.3 uM) very similar to those previously reported on
the authentic wild-type enzyme (24.2 p.M) while Kcat of the STI/
A protease has a 3.5-fold enhancements [25]. Here we further
measured the Kd value of the STI/A dimerization by ITC to be
13.4 uM (Figure lb), only slightly smaller than 21.4 uM for the
authentic WT protease [25], indicating that the STI/A mutation
only has a very small enhancement of the dimerization.
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Dynamical Enhancement of SARS-CoV 3CLpro
Equivalent monomer concentration
(mM)
Figure 1. Enzymatic activity and dissociation constant of the dimer-monomer equilibrium, (a). Enzymatic activity of STI/A vs. substrate
concentrations as measured by monitoring the increase of the emission fluorescence intensity at a wavelength of 538 nm. The Km and kcat values
are presented, (b). The ITC dilution profiles for measuring the dissociation constant of the dimer-monomer equilibrium for STI/A. The Kd and AH
values were obtained by fitting the ITC data with the built-in Microcal ORIGIN software, (c). Dimeric structure of the SARS 3CLpro with the catalytic
folds colored in blue (protomer 1) and cyan (protomer 2); the extra domains in red (protomer 1) and lightpink (protomer 2), as well as the connecting
loops in black (protomer 1) and purple (protomer 2). The catalytic dyad residue His41 is displayed as green sphere while Cys145 as yellow sphere.
Mutation residues S284T285I286 and N214 are displayed as spheres and the N-finger residues Ser1-Fhe8 are displayed as dots.
doi:1 0.1 371 /journal.pone.01 01 941 .g001
Crystal structure of the STI/A enzyme
To gain insights into the structural consequence of the STI/A
mutation, we determined its crystal structure at a resolution of
2.25 A in C2 space group, with one molecule in the asymmetric
unit. The R wor k factor of the final models for STI/A mutant was
18.4%, while the R fr( . e factor was 23.1%. Details of the data
collection and refinement statistics are presented in Table SI.
In the electron density map of the STI/A mutant, all 306
residues are visible and it adopts the classic dimeric structure with
the same packing of the two protomers as observed in all
previously-determined dimeric structures of the coronavirus
3CLpro [1,2,4,18,19,25,40-45]. The dimeric STI/A structure is
highly similar to those of the WT protease previously reported
(Figure 2a). If compared to the WT crystal structure (2H2Z) [18]
also determined at pH 6.0, with the authentic sequence in the
same C2 space group [18], the dimeric RMS deviations are 0.86
and 0.77 A respectively for the heavy (C, O, S and N) and
backbone atoms (C, Ca, N (backbone-amide), carbonyl). Even if
comparing at the individual protomer level, the RMS deviations
reduce to 0.55 and 0.49 A respectively for the heavy and backbone
atoms, implying that the packing of two protomers slightly differs
in the STI/A proteases. In the STI/A mutant, even for the key
residues constituting the catalytic machinery including the
catalytic dyad His41-Cysl45, oxyanion-loop Phel40-Cysl45,
Hisl63 and Glul66 critical for binding substrates; and Phel40,
His 172 in holding the substrate binding-pocket open, their
backbones and side-chains are almost superimposable to those in
the WT structure (Figure 2b). This observation implies that the
enhancement of the STI/A activity cannot be readily rationalized
only by the static structure. Furthermore, the mutation residues
Ser284-Thr285-Ile286 are located on the extra domain and far
away from the catalytic dyad His41-Cysl45 (Figure lc). We also
examined the B-factors of STI/A and WT proteases but it appears
extremely challenging to establish any precise correlation between
the B-factors and catalytic activity. Therefore, the catalytic
enhancement is most likely due to the change of the protein
dynamics of the enzyme induced by the mutations as we
previously demonstrated on the N214A mutant [25].
Intriguingly, the enzymatic activity of the wild-type 3CLpro has
been previously found to be pH-dependent where the optimum
activity is at pH 7.6 and the activity become much lower at
pH 6.0 [4,5,18,44—45]. Strikingly, upon pH changes, the extra
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Dynamical Enhancement of SARS-CoV 3CLpro
Catalytic^
Dyad V
Figure 2. Crystal structure of the STI/A mutant, (a). Overall superimposition of the dimeric STI/A (violet) and WT (cyan; PDB code of 2H2Z (18)
structures, (b) Superimpositions of the catalytically critical residues of STI/A (violet) and WT (cyan; PDB code of 2H2Z (18). (c). Diagram showing the
distance (d) between the mass centers of two extra domains; and the angle (0) by the mass centers of two extra domains as well as the mass center
of two chymotrypsin folds together (see Material and Methods for more details). The cavity volumes (V) of the nano-channels of STI/A (d) and WT (e)
as represented by violet dots, which were calculated with the program: POVME (38).
doi:1 0.1 371 /journal.pone.01 01 941 .g002
domains in the dimeric enzyme appear to undergo a 'rigid body
rotation/movement'. Inspired by this, here we calculated the
distances (d) between the centers of mass of two extra domains, as
well as the angles (©) formed by two mass centers of each extra
domains and the mass center of the two catalytic folds together
(Figure 2c), which are 32.4 A and 63.2 degree for the STI/A
determined here at pH 6.0 (3EA7), 33.7 A and 66.3 degree for the
wild-type enzyme at pH 6.0 (2H2Z) [18], 32.6 A and 63.3 degree
for the wild-type enzyme at pH 7.6 (1UK3), and 32.7 A and 63.7
degree for the wild-type enzyme at pH 8.0 (1UK2). This
observation implies that the slightly tighter packing of the two
extra domains is indeed associated with the higher catalytic
activity.
Previously, based on a systematic mutagenesis study, we have
proposed a nano-channel to be responsible for relaying the
mutation effect from the extra domain to the catalytic machinery,
which are mainly constituted by residues next to S284-T285-I286
on the extra domains as well as N-ffnger residues on the catalytic
folds [16]. Because the mutation of STI to Ala with a small side
chain results in a cavity, in STI/A the two extra domains come
slightly closer as described above, and consequently the volume of
the nano-channel (V) is 1293 A 3 in STI/A (Figure 2d), slightly
smaUer than that (1435 A 3 ) in WT (Figure 2e).
Molecular dynamics (MD) simulations
Molecular dynamics simulation is a powerful tool to provide
insights into the dynamic mechanism that underlies protein
function [25,46-50]. Therefore, we conducted 100-ns MD
simulations for the wild-type (2H2Z) [18] and our present STI/
A (3EA7) structures, both of which have been determined at
pH 6.0 and in crystals of the space group C2.
Figure 3 presents the root-mean-square deviation (RMSD) of
Cot atoms (from their positions in the energy minimized structures)
for the dimeric WT and STI/A (a-c), their catalytic folds (domains
I and II; d-f) and extra domains only (domain III, g-j).
Interestingly, in the context of the dimeric forms, STI/ A showed
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Dynamical Enhancement of SARS-CoV 3CLpro
larger overall RMSD than WT, with average RMSD values over
100-ns simulations of 2.64±0.32, 1.90±0.26 and 2.68±0.42 A for
three separate simulations of STI/A; and 1.60±0.26, 1.66±0.31
and 1.63±0.25 A for three simulations of WT. However, the
averaged Cot RMSD values of the chymotrypsin folds (Domain I
and II) of STI/A are only slightly larger than those of WT
(Figures 3d-3f), with average RMSD values over 100-ns simula-
tions of 1.40±0.21, 1.20±0.16 and 1.44±0.26 A for three
simulations of STI/A; and of 1.28±0.16, 1.17+0.17 and
1 .25 ±0.15 A for three simulations of WT. On the other hand,
the extra domains of STI/A have dramatically higher fluctuations
than WT in simulations as indicated by its higher RMSD values
(Figures 3g-3i), with average RMSD values over 100-ns simula-
tions of 3.57±0.54, 2.19±0.53 and 3.26±0.86 A for three
simulations of STI/A and of 1.75±0.51, 1.62±0.31 and
1.61 ±0.31 A for three simulations of WT. These results clearly
demonstrate that the larger RMSD values for the dimeric STI/A
are mostiy resulting from the motions of the extra domains. This
premise is further evident from the root-mean-square fluctuation
(RMSF) averaged over 100 ns for both STI/A and WT residues
(Figures 3j-3o).
Indeed, for STI/A, the distance d (Figures 4a-c) and angle ©
(Figure 4d-f) as defined in Figure 2d have further reduced after
~10 ns in simulations 1 and 3 and after ~45 ns in simulation 2.
For the average distance over 100-ns trajectories, STI/A has
28.77±0.90, 30.69± 1.16 and 29.20± 1.29 A respectively for three
simulations, while WT has 32.78±1.07, 34.12±0.62 and
33.33±0.656 A respectively. For the average angle over 100-ns
trajectories, STI/A has 54.78± 1.781, 58.98±2.544 and
55.25±2.535 degree respectively for three simulations, while
WT has 64.54± 2.250, 66. 65 ±1.461 and 65.43 ±1.614 degree
respectively. These results indicate that in all three separate
simulations, the two extra domains of STI/A become more tightly
packed than those of WT. Consequently the volumes of the nano-
channel significantly reduced for STI/ A in all three simulations as
compared to those of WT (Figures 4g-h), with average values of
512.62±212.52, 890.26±247.17 and 579.80±261.51 A 3 respec-
tively for three simulations of STI/A, and of 1204.35±255.69,
1428.93± 121.50 and 1319.94± 143.21 A' 5 respectively for WT.
f (b) M (c) ]
_l i i i i I i i i i L
_l i i i i I i i i i L
Residue
Figure 3. Overall dynamic behaviors in the W1D simulations. Root-mean-square deviations (RMSD) of the Cot atoms (from their positions in the
energy minimized structures) for three independent MD simulations of the dimeric STI/A (black) and WT (red) for the whole enzyme (a-c); catalytic
fold (d-f); and extra domain (g-i). Root-mean-square fluctuations of the Ca atoms computed for three independent simulations for protomer A (j-l)
and protomer B (m-o) of STI/A (black) and WT (red). Protomer A and B are denoted as PI and P2 respectively.
doi:1 0.1 371 /journal.pone.01 01 941 .g003
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Dynamical Enhancement of SARS-CoV 3CLpro
Time (ns)
Figure 4. Changes of the packing interaction between two extra domains. Three separate time-trajectories of the distance d (a-c); angle 0
(see Figure 2d) (d-f) and volume of nano-channel (residue 284-286), V (g-i) of STI/A (black) and WT (red).
doi:1 0.1 371 /journal.pone.01 01 941 .g004
We have also calculated the hydrogen bond occupancy of all
simulations for both STI/A and WT, and Table 1 presents the
hydrogen bond occupancies with the difference >5% between
STI/A and WT. Interestingly, although the STI/A mutations are
on the extra domain, only limited intra-domain hydrogen bonds
show significant occupancy differences within the extra domain
(Table 1). This suggests that similar to the tighter packing between
two extra domains triggered by the high pH in WT [44] , here the
tighter packing induced by the STI/A mutations is also largely
resulting from the 'rigid body rotation/movement' of the extra
domains without significantly affecting the packing within the
extra domains. Furthermore, the slight dynamic changes over the
extra domains can also be transmitted to the catalytic fold, as
exemplified by the significant changes of hydrogen bond
occupancies between Asp295 and Thrill (Table 1).
Dynamic behavior of the catalytic dyad and oxyanion
hole
In the previously-determined crystal structures of SARS
3CLpro, the distance between NE2 of His41 and SG of Cysl45
ranges from 3.6 to 3.9 A. Furthermore, all previous MD
simulations revealed that the dynamic stability of this distance is
extremely critical for the stable formation of a hydrogen bond,
which appears to be pivotal for maintaining the catalytic
competency of the SARS 3CLpro [10,23,25,51-53]. Also for the
active WT enzyme, this distance has been previously demonstrat-
ed to be dynamically stable in MD simulations. Indeed, as in our
previous simulations, the distances of both protomers of the WT
were found to be dynamically stable for all 30 ns while those of the
inactive N214A mutant became unstable and larger despite
sharing a superimposable crystal structure to the WT [25]. In our
current MD simulations of 100 ns, the distances between NE2 of
His41 and SG of Cysl45 of STI/A are: 3.80±0.60 A, 3.91±0.51
and 3.78±0.50 A respectively for three simulations of the
protomer 1 (Figures 5a-5c), and 3.91 ±0.48, 3.87±0.45 and
3.86±0.48 A respectively for the protomer 2 (Figures 5d-5f). In
contrast, the distances in WT are: 3.82±0.46, 3.83±0.49 and
4.07 ±0.76 A respectively for three simulations of the protomer 1,
and 4.08±0.52, 3.84±0.49 and 3.89±0.43 A respectively for the
protomer 2. As a consequence, the distance of STI/A ranges from
3.78 to 3.91 A while WT from 3.82 to 4.08 A, suggesting that
overall this distance is more dynamically stable in STI than that in
WT. In particular, after 80 ns, in simulation 3 of the WT
protomer 1, this distance becomes largely fluctuating (Figure 5c),
while for STI/A, no such large fluctuation can be observed for all
100 ns. The Chi2 angles of His41 have similar dynamical
behaviors (Figures 5g-51) and the large fluctuation is also observed
(Figure 5i).
One key component of the catalytic machinery of the SARS
3CLp is the substrate-binding pocket composed of six substrate
sites, namely S1-S6, which correspond to the P1-P6 residues of
the substrate [1,2,4,23,25]. The SI substrate site is the most critical
as it confers an absolute specificity for a Gin at the PI position of
the substrate. As such, maintaining the intact conformation of SI
substrate site is especially vital for catalysis. Briefly, the SI
substrate site can be divided into four parts: the oxyanion hole;
Hisl63; Glul66; Phel40 and its stabilizing elements [1,2,4,23,25].
The oxyanion hole refers to a structural element to donate two
hydrogen bonds from main-chain amides of Gly- 1 43 and Cys- 145
to accommodate the main-chain oxygen of Gln-P 1 as well as the
tetrahedral intermediate during catalysis. Previously we demon-
strated that in the inactive monomer R298A, the most
distinguishable change is the collapse of the oxyanion hole as
triggered by the chameleon formation of a short 3 10 -helix from a
loop over residues Serl39-Phel40-Leul41 [22]. Remarkably, in
our previous MD simulations, residues Ser 1 39-Phe 1 40-Leu 1 4 1 of
the WT maintained an extended active conformation while those
of N2 1 4A frequently jumped to sample the 3 1 0 -helix conformation
characteristic of the collapsed oxyanion hole [25] .
In the present simulations of both STA and WT up to 100 ns,
the three residues have similar dynamical behaviors in their
backbone conformations (Figure SI), as well as the similar
hydrophobic stacking interactions among Phel40, His 163 and
His 172.
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Dynamical Enhancement of SARS-CoV 3CLpro
Table 1. Hydrogen Bond Occupancy for STI/A and WT with Significant Differences.
Donor
Acceptor
Average (%)
Res No.
Name
Atom
Chain
Res No.
Name
Atom
Chain
STI/A
WT
(STI-WT)
Extra Domain
276
MET
N
A
271
LEU
O
A
0.0
5.5
-5.5
276
MET
N
B
271
LEU
O
B
0.2
10.7
-10.5
278
GLY
N
B
285
THR
0
B
0.0
13.9
-13.9
230
PHE
N
A
226
THR
0
A
62.4
78.7
-16.3
230
PHE
N
B
226
THR
0
B
67.1
82.5
-15.4
257
THR
N
B
253
LEU
0
B
47.1
59.3
-12.2
299
GLN
N
A
296
VAL
0
A
13.1
36.1
-23
299
GLN
N
B
296
VAL
0
B
29.1
40.0
-10.9
-13.5 + 5.1
Extra Domain-Chymotrypsin Fold
111
THR
OG1
A
295
ASP
OD1
A
30.8
0.1
30.7
111
THR
OG1
B
295
ASP
OD1
B
34.3
15.7
18.6
24.7 ±8.6
N-Fingers
4
ARG
NH1
A
290
GLU
OE2
B
29.3
44.6
-15.3
4
ARG
NH1
B
290
GLU
OE2
A
31.0
54.6
-23.6
4
ARG
NH2
A
137
LYS
O
B
14.1
24.4
-10.3
4
ARG
NH2
B
137
LYS
O
A
5.8
22.9
-17.1
5
LYS
NZ
A
288
GLU
OE2
A
31.6
20.6
11.9
5
LYS
NZ
B
288
GLU
OE2
B
21.3
14.0
7.3
7
ALA
N
A
125
VAL
O
B
83.6
67.1
16.5
-4.5+15.8
Helix A at Domain 1
10
SER
N
A
10
SER
OG
B
71.3
34.3
37.0
10
SER
N
B
10
SER
OG
A
61.5
33.8
27.7
11
GLY
N
B
14
GLU
OE1
A
93.3
32.8
60.5
11
GLY
N
A
14
GLU
OE1
B
62.4
72.8
-10.4
13
VAL
N
A
10
SER
O
A
87.6
48.1
39.5
15
GLY
N
B
11
GLY
O
B
53.2
9.1
44.1
95
ASN
ND2
A
15
GLY
O
A
72.7
67.0
5.7
95
ASN
ND2
B
15
GLY
O
B
75.6
63.9
11.7
27.0±23.2
Asn28
28
ASN
ND2
A
117
CYS
O
A
27.5
10.4
17.1
28
ASN
ND2
B
117
CYS
O
B
32.4
18.0
14.4
28
ASN
ND2
A
145
CYS
O
A
87.6
30.5
57.1
28
ASN
ND2
B
145
CYS
O
B
78.0
56.0
22.0
27.7±19.9
Glu166
172
HIE
NE2
A
166
GLU
OE2
A
43.3
19.6
23.7
Linker between Catalytic and Extra Domains
175
THR
OG1
A
176
ASP
O
A
16.9
26.5
-9.6
175
THR
OG1
B
176
ASP
O
B
27.2
39.2
-12.0
105
ARG
NH1
A
180
LYS
O
A
14.9
25.1
-10.2
105
ARG
NH1
B
180
LYS
O
B
12.1
34.3
-22.2
182
TYR
N
A
174
GLY
O
A
88.9
96.4
-7.5
CO
TYR
N
B
174
GLY
O
B
89.4
95.6
-6.2
-11.3 + 5.7
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Dynamical Enhancement of SARS-CoV 3CLpro
Table 1. Cont.
Donor
Acceptor
Average (%)
Res No.
Name
Atom
Chain
Res No.
Name
Atom
Chain
STI/A
WT
(STI-WT)
Chymotrypsin Fold
19
GLN
NE2
A
26
THR
OG1
A
36.3
21.5
14.8
19
GLN
NE2
B
26
THR
OG1
B
59.5
0.0
59.5
19
GLN
NE2
A
119
ASN
0
A
5.5
20.3
-14.8
19
GLN
NE2
B
119
ASN
0
B
2.1
11.3
-9.2
21
THR
OG1
A
25
THR
0
A
0.0
8.8
-8.8
21
THR
OG1
B
25
THR
0
B
0.0
18.2
-18.2
22
CYS
N
A
25
THR
0
A
98.2
65.6
32.6
22
CYS
N
B
25
THR
0
B
98.5
63.1
35.4
25
THR
OG1
A
44
CYS
0
A
85.9
40.8
45.1
25
THR
OG1
B
44
CYS
0
B
75.9
52.8
23.1
25
THR
N
A
22
CYS
0
A
47.8
34.6
13.2
25
THR
N
B
22
CYS
0
B
43.4
24.4
19.0
26
THR
OG1
A
21
THR
OG1
A
74.4
62.3
12,1
26
THR
OG1
B
21
THR
OG1
B
85.0
25.8
59/2
113
SER
OG
A
127
GLN
OE1
A
95.5
84.4
11.1
113
SER
OG
B
127
GLN
OE1
B
92.7
46.1
46.6
116
ALA
N
A
124
GLY
0
A
92.7
75.6
17.1
117
CYS
N
B
147
SER
OG
B
80.2
65.8
14.4
121
SER
N
A
118
TYR
0
A
65.1
49.1
16.0
121
SER
N
B
118
TYR
0
B
61.6
56.0
5.6
135
THR
N
A
133
ASN
OD1
A
63.6
55.5
8.1
135
THR
N
B
133
ASN
OD1
B
61.6
54.7
6.9
148
VAL
N
A
115
LEU
0
A
63.0
20.8
42.2
-18.7±21.8
doi:1 0.1 371 /journal.pone.01 01 941 .t001
Dynamic behavior of Hisl 63 and Glu166
The Glu-Pl substrate binding site includes essential substrate
recognition residues: Hisl63, Glul66 and Hisl72. Extensive
studies previously revealed that increased side-chain flexibility of
His 163 and Glul66 decreases the enzymatic activity [44], and
mutation of Glul66 to alanine reduces the enzymatic activity by
many folds [54]. Particularly these three residues were found to
play a key role in the pH-dependent enhancement of the catalytic
activity by both experiments and simulations [44]: the reduced
contact between His 163 and Glul66 allowed Glul66 to engage in
hydrogen bonding with Hisl 72 and consequently adopted optimal
conformations for substrate-binding at pH 7.6. In contrast, the
increased contact between His 163 and Glul66 decreased hydro-
gen bond between Glul66 and Hisl 72, and thus reduced the
stability of the substrate pocket at pH 6 [44] .
In our current 100-ns MD simulation, the backbone confor-
mations of both His 163 and Glul66 are indistinguishable between
STI/A and WT. In STI/A, the distances of Hisl63-Glul66 are:
7.97±0.32, 7.75±0.35 and 7.89±0.35 A (with the average of
7.87 A) for three simulations of protomer 1; 7.60±0.56,
8.31 ±0.31 and 8.18±0.50 A (with the average of 8.03 A) for
protomer 2. In WT, the distances are: 8.02±0.42, 7.94±0.37 and
8.17±0.47 A (with the average of 7.86 A) for three simulations of
protomer 1; 7.72±0.56, 8.05±0.34 and 7.81±0.44 A (with the
average of 8.04 A) for protomer 2. Furthermore, the dynamic
behavior of the Hisl63-Hisl 72 distance is highly similar in both
STI/A and WT (Figure S2a-S2f).
Interestingly, STI/ A showed enhanced dynamic stability in the
Glul66 side-chain conformation (Figures 6a-6f) and smaller
centroid distance between the side chains of Glul66 and His 172
(Figures 6g-61). The averaged centroid distances of Glul66 and
His 172 are: 4.80 A and 4.81 A respectively for two protomers of
STI/A; and 4.73 and 5.13 A respectively for two protomers of
WT. The slight decrease in the distance between Glul66 and
His 172 in STI/A leads to an increased hydrogen bond occupancy
between Glul66 and Hisl72: 43.3% in STI/A versus 19.6% in
WT (Table 1). Previously, the molecular mechanism for the pH-
triggered activity enhancement has also been largely attributed to
the increase in the Glul66 dynamic stability, which results from
the different protonation states of Hisl63 and Hisl72 at higher pH
[43] . This implies that the activity enhancement triggered by high
pH and STI/A mutation may share overlapped allosteric
pathways.
Dynamic behavior of N-finger and a-helix A residues
In the dimeric structures, the N-finger residues Ser 1 -Ala7 of one
protomer have extensive contacts with residues of another
protomer which include those constituting the substrate-binding
and the catalytic pocket as well as critical for the dimerization. On
the other hand, the N-finger residues also make extensive contacts
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9
July 2014 | Volume 9 | Issue 7 | e101941
Dynamical Enhancement of SARS-CoV 3CLpro
Time (ns)
Figure 5. Dynamic behavior of the catalytic dyad. Three separate time-trajectories of the distance between NE2 of His41 and SG of Cys145
atoms of protomer A (a-c) and protomer B (d-f) for STI/A (black) and WT (red). Three separate time-trajectories of the Chi2 dihedral angle of His41 of
protomer A (g-i) and protomer B (j-l) for STI/A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
doi:1 0.1 371 /journal.pone.01 01 941 .g005
with the extra domains and are key component of the nano-
channel [16]. As such, the dynamical changes of the extra domains
may be relayed to other catalytic domain residues via the N-finger
residues.
In the current MD simulations, the backbone conformations of
the N-linger residues including Arg4 and Lys5 are very similar in
both STI/A and WT. Nevertheless, the Arg4 side-chain confor-
mations indicated by Chil is lopsided in STI/A: Arg4 sampled
either of the two conformation clusters (30°<Chil<90° and —
30°<Chil< — 90°) in protomer 1 (Figures S3a-S3c) but sampled
consistently only one conformation cluster (— 30°<Chil< — 90°) in
protomer 2 (Figures S3d-S3f). On the other hand, Arg4 in both
protomers of WT sampled 3 conformation clusters. The side-chain
conformation (Chil) of Lys5 in STI/A prefers 2 conformation
clusters (- 180°<Chil<- 150° and 150°<Chil<180°) (Figures
S3m-S3r) while Lys5 in WT is restricted to one conformation
cluster (— 90°<Chil< — 30°). Interestingly, as shown in Table 1, in
STI/A, Arg4 has lower hydrogen bond occupancies with Glu290
and Lysl37, while Lys5 has higher hydrogen bond occupancies
with Glu288.
The slight dynamical changes over the N-finger residues appear
to affect the downstream helix A (Serl0-Glyl5), which are
mutually aligned at the dimer interface and engaged in inter-
protomer hydrogen bonds and salt-bridges. Previously, the
mutation of Glyl 1 to Ala has been shown to completely abolish
the dimeric structure and lead to a collapsed catalytic machinery
[23]. In the present simulations, although the backbone confor-
mations of the helix A (Serl0-Glyl5) are largely similar in the
simulations of both STI/A and WT (Figure S4), the dynamic
stability of the helix is largely enhanced, as evidenced by the
significantly increased occupancies of the intra-residue SerlO
hydrogen bonds and those between Glyll and Glul4, SerlO and
Vall3, Glyll-Glyl5, as well as Glyl5-Asn95 (Table 1).
Increased conformational stability of Asn28 and Thr25
Previously, Asn28 has been identified to be a key component to
mediate both catalysis and dimerization of the SARS 3CLpro via a
long-range interaction network [7,8,21]. More specifically, it
maintains the conformation integrity of and positioning of Cys 1 45
(catalytic residue), Lysl37-Phel40 (part of the oxyanion loop),
Tyr 1 26 and Cys 1 1 7 through a hydrogen bond network composed
of Asn28-Cysl45, Asn28-Glyl43, Asn28-Cysll7 and Asn28-
Glyl20. Indeed, the mutation of Asn28 also led to a complete
elimination of the dimeric structure and an inactivated and
collapsed catalytic machinery [21]. In the present simulations, the
backbone conformations of Asn28 are largely similar in STI/A
and WT. However, the side-chain conformations of Asn28 as
reflected by Chil (Figures 7a-7f) and Chi2 (Figures 7g-71) exhibited
an enhanced conformational stability in STI/A. On the other
hand, in STI/A, Asn28 has significantly increased occupancies of
the hydrogen bonds with Cysl 1 7 and Cysl45 (Table 1). It appears
that the enhanced stability of Asn28 contributes to the dynamical
stability of the catalytic dyad (His41 and Cys 145) (Figure 5).
Furthermore, we also observed an increased dynamical stability
for residues of the Leu-P2 substrate pocket in the STI/A
simulations. The Leu-P2 substrate binding site consists of Thr25,
Leu27, Val42, Cys44, Thr47, Asp48, Met49, Tyr54, Leul64, and
Met 165 and these residues form a hydrophobic pocket that is
receptive to a bulky side-chain such as Leu, Phe and Val [52].
Studies indicated that the physical dimensions or/and specific
conformation of Leu-P2 substrate site determined both substrate
binding specificity and consequentially the enzymatic catalytic
rate. Thr25 and Cys44 lie at the edge of the Leu-P2 substrate
binding site. The backbone and side-chain conformations of Cys44
are largely similar in both STI/A and WT simulations.
Interestingly Thr25 shows a slighdy enhanced dynamical stability
of the backbone (Figures 8a-81). Furthermore, the distance
(Figures 8m-8r) and volume enclosed (Figures 8s-8x) between
Thr25 and Cys44 have enhanced dynamical stability profiles in
STI/A simulation: the averaged distance between Thr25/OGl
and Cys44/0 is: 2.87±0.36 A (protomer 1), 3.05±0.54 A
(protomer 2) for STI/A; and 3.63±0.92 A (protomer 1),
3.40±1.00 A (protomer 1) for WT. On the other hand, there is
an approximate 2-fold increase in hydrogen bond occupancy
between Thr25/OGl and Cys44/0 in STI/A simulations
(Table 1). The significant fluctuations of the distance and volume
between Thr25 and Cys44 in WT may result in an increased
insertion of the methyl moiety of Thr25 side-chain into the cavity
of Leu-P2 substrate site, thus impeding the docking of substrate
into the substrate pocket or reduce the enzyme grip on the
substrate during enzymatic catalysis.
Other regions
We have also compared the dynamic behaviors of all other
residues in STI/A and WT and found them to be very similar.
Interestingly, although the STI/A mutations are on the extra
domains, many residues in the chymotrypsin fold have the
occupancies of their intra-domain hydrogen bond significandy
affected (Table 1). First, the linker residues connecting the catalytic
and extra domains have reduced hydrogen bond occupancies,
which come from the rearrangement of the orientation between
the catalytic and extra domains via 'rigid body rotation/
movement' in SIT/ A. Second, in STI/A simulations, except for
the hydrogen bonds of Glnl9-Asnll9 and Thr21-Thr25 which
PLOS ONE | www.plosone.org
10
July 2014 | Volume 9 | Issue 7 | e101941
Dynamical Enhancement of SARS-CoV 3CLpro
o Si
D) 0
0)
"Q -90
CD
LU
T~~ ID
UJ Q 0
(a)
(b)
(g)
(h)
! i»
■ (C) !
ISO
30
0
-3D
m 1 V 1 '.
■(e) ! i \
50
MI
50 100
50 100
6
m
) 50 1
0
□
•03
50 1
0
"(k)
m M r h i
tf) . IN
in
Time (ns)
Figure 6. Dynamic behavior of the Glu166-His172 interaction. Three separate time-trajectories of the Chi1 dihedral angle of Glu166 of
protomer A (a-c) and protomer B (d-f) for STI/A (black) and WT (red). Three separate time-trajectories of the distance between Glu166 and Hisl 72 of
protomer A (g-i) and protomer B (j-l) for STI/A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
doi:1 0.1 371 /journal.pone.01 01 941 .g006
have reduced occupancies, hydrogen bonds of other catalytic fold
residues have significantly increased occupancies. These observa-
tions clearly indicate that the effects triggered by the STI/A
mutations can be indeed transmitted to the catalytic fold and
enhance the dynamic stability of the catalytic machinery, which
ultimately leads to the increased catalytic activity of the STI/A
mutant.
Correlation analysis
As seen in the mutual information profiles (Figure 9), in the
protomer 1 of WT, fragments of both catalytic and extra domains
have highly correlated motions, which include Phe3-Ser62
contaning the N-finger, helix A, Thr25, Asn28, Cys44 and
catalytic dyad residue His41; Leul 15-Cysl56 containing CII-BII
residues, oxyanion loop Serl39-Leul41, and catalytic dyad
residue Cysl45; Vall86-Thrl98 within the loop connecting the
catalytic and extra domain; Asn214-Asn238 containing Asn214;
and Ile281-Phe305 containing S284-T285-I286. These fragments
cover all residues which have been identified to be critical for
dimerization and catalysis previously as well as in the present
study, which constitute a correlated network over the whole
protease (Figure 9). Interestingly, here the loop residues Vail 86-
Thrl98 are revealed to be a key component of this network.
Previously their role in dimerization and catalysis has been mosdy
unknown and thus it is worthwhile to experimentally characterize
in the further. Furthermore, although the correlation pattern in
the protomer 2 of WT remains largely similar, the significandy
correlated pairs of residues slightly changed, thus leading to the
less correlation of some catalytic domain residues (Figure 9).
Strikingly, although the N2 1 4A mutation significantly provoked
the dynamics of the whole protease [25], the mutual information
profiles reveal that it globally decouples the correlation of the
paired residue motions (Figure 9). As a consequence, the first half
of the catalytic fold which hosts His41, one of the catalytic dyad
residues, loses the significant correlation to the rest of the protease
in both protomers. The results on the correlation analysis of both
WT and N2 1 4A strongly suggest that the networks to correlate the
motions over the whole protease are essential for implementing its
catalytic action, and the dynamic perturbation onto a key
component even without detectable conformational change is
sufficient to dramatically inactivate the catalytic machinery by
disrupting the correlation network. On the other hand, for STI/A,
the difference of the mutual information profiles between STI/A
and WT is relatively small. In the protomer 1 of STI/ A, although
the correlated motions of some pairs of residues become less
significant as compare to those in WT, the correlated motions
become significantly increased for residues Gly2-Arg4 with many
other residues distributed over the whole protease. In the second
protomer of STI/A, the correlated motions over the majority of
the residue pairs become enhanced as compared to those of WT.
As such, it is not straightforward to attribute these changes to the
activity enhancement in STI/A. It appears that unlike the N214A
mutation which decouples the global correlated motions, thus
inactivating the catalytic machinery, the STI/A mutation
enhances the catalytic activity by the specific allosteric pathways
which manifest as the altered correlation patterns.
P1
00 T" .90
CM £
^ o m
(/) S m
£ 90
^ Ol
4 0
CS -90
O 180
(b)
IT
(c)
(d)
(g)
(h)
(i)
am
a)
Time (ns)
P2
(e)
(f)
(k)
50 100
50 100
Figure 7. Dynamic behavior of Asn28. Three separate time-trajectories of the Chi 1 dihedral angle of Asn28 of protomer A (a-c) and protomer B
(d-f) for STI/A (black) and WT (red). Three separate time-trajectories of the Chi2 dihedral angle of Asn28 of protomer A (g-i) and protomer B (j-l) for STI/
A (black) and WT (red). Protomer A and B are denoted as P1 and P2 respectively.
doi:1 0.1 371 /journal.pone.01 01 941 .g007
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Figure 8. Dynamic behavior of Thr25 and Cys44. Three separate time-trajectories of Phi (a-c) and Psi (d-f) dihedral angles of Thr25 of protomer
A; and Phi (g-i) and Psi (j-l) dihedral angles of Thr25 of protomer B for STI/A (black) and WT (red). Three separate time-trajectories of the distance
between Thr25 and Cys44 of protomer A (m-o) and protomer B (p-r) for STI/A (black) and WT (red). Three separate time-trajectories of the volume
enclosed by Thr25 and Cys44 of protomer A (s-u) and protomer B (v-x) for STI/A (black) and WT (red). Protomer A and B are denoted as PI and P2
respectively.
doi:1 0.1 371 /journal.pone.01 01 941 .g008
Discussion
Due to the severity of the worldwide SARS epidemic, the
SARS-CoV received extremely-intense research efforts worldwide
immediately after its outbreak. The SARS 3CLpro not only
represents a promising target for developing therapeutic agents,
but also serves as an excellent model for understanding how the
evolutionarily-acquired non-catalytic domains mediate the enzy-
matic mechanisms. Previously, it has been found that by acquiring
additional non-catalytic domains during evolution, many enzymes
gain altered catalytic mechanisms or/ and be connected to cellular
signaling networks [55-56]. Indeed, upon acquiring the C-
terminal extra domain, SARS 3CLpro suddenly needs the
dimerization to activate its catalytic machinery. Previous studies
have uncovered that the monomeric structures of the SARS
3CLpro have the same collapsed catalytic machinery, regardless of
being triggered by G11A, N28A or S139A mutations on the
catalytic domains [21,23,24], or by R298A on the extra domain
[22]. This suggests that the dimerization is commonly controlled
by a structurally-allosteric network composed of residues of both
catalytic and extra domains. With MD simulations, we have
shown that the collapsed catalytic machinery was not only
structurally-distinguishable from, but also dynamically well-sepa-
rated from the activated state of SARS 3CLpro [25] . Remarkably,
despite having almost the same three-dimensional structure as
WT, in MD simulations, the dimeric but inactive N2 1 4A mutant
frequently jumped to sample the conformations of the collapsed
state, thus leading to the proposal of the "dynamically-driven
inactivation" for the catalytic machinery of SARS 3CLpro [25].
This observation implies that the mutation effect of N2 1 4A has
been relayed to inactivating the catalytic machinery through the
dynamic allostery.
Here we studied another mutant STI/A with mutations on the
extra domain, which has a significandy-increased activity but only
slightly-enhanced dimerization. The successful determination of its
crystal structure indicates that STI/A still adopts the dimeric
structure almost identical to that of WT, only with slightly tighter
packing between two extra-domains due to the cavity created from
replacing Ser284-Thr285-Ile286 with Ala (Figure 2a). In partic-
ular, the key residues constituting the catalytic machinery are
almost superimposable to those of WT (Figure 2b). We thus
hypothesized that the activity enhancement may be mosdy
resulting from the dynamic allostery, as we previously observed
in the N214A mutant [25]. Consequendy we conducted 100-ns
MD simulations for both STI/A and WT, which represent the
longest simulations reported for 3CLpro so far. An exhaustive
analysis of MD results revealed that STI/ A and WT have very
similar dynamical behaviors for most residues. Nevertheless, in the
MD simulations, STI/A did show some dynamical behaviors
different from those in WT. The most significant change in STI/A
is that in the simulations the two extra domains became further
tightly packed in STI/A, mosdy driven by the hydrophobic
interactions by Ala284-Ala285-Ala286-Leu287. This led to a
dramatic reduction of the volume of a nano-channel constituted by
residues of both catalytic and extra domains (Figures 10a- lOe),
which we previously proposed to relay the perturbation on the
extra domains to the catalytic machinery [16]. The volume
reduction in the nano-channel triggered slight changes of
dynamics of some nano-channel residues (Figure 1 Of), in particular
the N-finger residues (Figure S3), which are also reflected by the
redistribution of the hydrogen bond occupancy (Table 1).
Interestingly the Helix A residues also become slightly more stable
as evident from their backbone conformations (Figure S4) and
increased hydrogen-bond occupancy for the residues SerlO and
Glyl 1 (Table 1). The dynamic changes over N-finger and helix A
appears to be further transmitted to residues over the BII and CII
beta-strands including Thrill, Serll3, Leul 15-Tyrl 18 and
Serl23 (Figure 1 Of), which ultimately lead to the enhanced
dynamic stability of residues constituting the catalytic machinery,
such as Asn28, Thr25 and Cys45; Glul66 and Hisl72; as well as
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Dynamical Enhancement of SARS-CoV 3CLpro
Figure 9. Networks of correlated motions in WT, STI/A and N214A. Mutual information matrixes calculated from the MD simulation data of
WT, STI/A and N214A by Mutlnf (39); as well as the SARS 3CL protease structures with residues having significant correlation motion displayed in
spheres which are colored in red if in the protomer 1; and brown in the protomer 2. The catalytic dyad residue His41 is displayed as green sphere
while Cys145 as yellow sphere. The STI/A and N214A mutation residues are colored in splitpea. Yellow boxes in WT highlight the highly correlated
motions between Phe3-Ser62 and: CII-BII (Leul 15-Cys156), oxyanion loop (Ser139-I_eu141), as well as extra domain (Asn214-Asn238, Ile281-Phe305).
Yellow boxes in PI of STI/A highlight the highly correlated motions of N-fingers with the other regions of the enzyme, similar to the WT while in P2 of
STI/A highlight the expanded, highly correlated motions between the Domain III (Asn214-Asn238, Ile281-Phe305) and: substrate pocket (LeuP1), CII-
BII (Leu115-Cys156), oxyanion loop (Ser139-Leu141), pocket 2 (Ser147-Thr175), loop region (Val186-Thr198). Yellow boxes in P1 of N214A highlight
the correlated motions amongst residues in internal (Ile59-Arg105), CII-BII (Leul 15-Cys156), oxyanion loop (Ser139-Leu141), catalytic dyad (Cys145)
and the A Helix (Ser10-Glu14); while in P2 of N214A highlight the correlated motions within Domain III.
doi:1 0.1 371 /journal.pone.01 01 941 .g009
the catalytic dyad His41-145. The enhanced dynamical stability of
these residues appears to be responsible for the increased activity
of STI/A. Indeed, previously high catalytic activity of some
enzymes has been correlated with their high stability [57-59] and
in particular, a lipase mutant with higher catalytic activity has
been characterized to have active sites of higher dynamical rigidity
by both MD simulation and experimental studies [59].
Amazingly, the residues involved in the dynamic transmission in
STI/A have been previously characterized to be also critical for
maintaining dimerization. For example, the N-finger [20], Glyl 1
[23] and Asn28 [21] residues are particularly important and
mutation/ deletion of them has been shown to abolish the dimeric
structures. This implies the existence of a correlated interaction
network, which is constituted by interactions of residues of both
catalytic and extra domains [21,22]. Indeed, our correlation
analysis by Mutlnf [39] reveals that such a global network of the
correlated motions does exist in the WT protease, whose
components include all residues identified to be critical for its
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Dynamical Enhancement of SARS-CoV 3CLpro
0 ns 25 ns 50 ns 75 ns 100 ns
Figure 10. Transmission of the STI/A mutation effects on the extra domains to the catalytic machinery by the dynamically-driven
allostery. (a)-(e). The cavity volumes of the nano-channel of STI/A in the first simulation at 0, 25, 50, 75 and 1 00 ns. (f). The crystal structure of STI/A
with key residues having relevant dynamical changes displayed and labeled. The cavity is represented by the violet mesh.
doi:1 0.1 371 /journal.pone.01 01 941 .g010
dimerization and catalysis previously as well as in the current study correlation of the network components while the STI/A mutation
(Figure 10). Most remarkably, the N214A mutation appears to enhance the activity by altering the correlation pattern. Indeed,
inactivate the catalytic machinery at least pardy by decoupling the the inactive N2 1 4A mutant does have a slighdy weakened
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Dynamical Enhancement of SARS-CoV 3CLpro
dimerization [25] while the more active STI/A mutant has a
slightly enhanced dimerization, which strongly supports the
proposal that the specific structured crowding can have significant
effects on enzymatic catalysis through mediating protein dynamics
and their correlations [49].
The results thus decipher a global correlation network in the
SARS 3CL protease which not only couples the dimerization and
catalysis by the structural allostery as previously demonstrated
[21-24], but also by the dynamic allostery. Previously, the
dynamic changes triggered by mutations have been extensively
demonstrated to mediate enzymatic catalysis by both experiments
and MD simulations [47-49,60-64] . However, it still remains rare
to find that the catalytic machinery can be dynamically modulated
by the mutations on the evolutionarily-gained non-catalytic
domain, which are also far away from the active center. To the
best of our knowledge, the SARS 3CLpro appears to be the first
example that without having significant structural change over the
active pocket, the mutation perturbations on the evolutionarily-
acquired non-catalytic domain can be relayed by the dynamic
allostery into manifesting opposite catalytic effects: inactivation of
catalysis in N214A and enhancement in STI/A. This proposition
implies that in addition to the structural allostery, the dynamic
allostery also plays key roles in controlling catalysis, which may
extensively exists in other enzymes.
New coronaviruses including human beta-coronavirus 2c
EMC/2012 (HCoV-EMC) may cause great threats to human
health in the near future [1,2,26]. However, one unsolved
challenge to fight against them is to design inhibitors for the
3CL proteases with high specificity. Based on the present results, a
promising avenue may be opened up to design very specific
inhibitors to disrupt the global networks of the correlated motions
through targeting the network components unique for each 3CL
protease.
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Figure SI Dynamic behavior of the oxyanion loop
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(TIF)
Figure S4 Dynamic behavior of the Helix A residues.
Ramachandran plots of the residues SerlO-Glyl 1-Lysl2-Vall3-
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(TIF)
Table SI Data collection and refinement statistics for
the STI/A mutant.
(DOCX)
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